Leader peptidase is an intracellular protease that plays an important role in protein export and catalyzes the removal of the amino-terminal leader sequences from exported proteins. The mechanism of action of leader peptidase is unknown and all available data suggests that it may belong to an unknown class of proteinase. Recently, our laboratory has provided evidence that suggest that leader peptidase is a member of an unusual class of serine protease that utilizes a lysine residue for catalysis. In this research proposal, we propose 1) to probe the catalytic mechanism of leader peptidase using kinetics, 2) to characterize an active delta2-75 leader peptidase fragment, 3) to test the serine/lysine catalytic model of leader peptidase, 4) to identify the substrate-binding residues within leader peptidase, 5) to map out the active site region, and 6) to prepare a fluorogenic pre-protein substrate for purposes of developing a rapid, continuous assay of leader peptidase activity. Oligonucleotide-directed mutagenesis, gene cloning, protein chemistry, kinetics, fluorescence, and CD spectroscopy techniques will be used to achieve these specific aims. The X-ray structure of the delta2-75 leader peptidase will be determined in a collaborative project. These studies will also help in the design of bacterial antibiotics that are targeted against leader peptidase, which is essential to bacterial growth.